Lecture 14

Question 1

describe endocytosis pathway - specific

Answer 1

• Vesicle traffic between PM, early endosome and trans-Golgi
• Early endosome matures into multivesicular body (MVB) and late endosome= Membrane switches
  from Rab5 to Rab7
• Late endosome matures into lysosome
• Other vesicles traffic to lysosome

Question 2

describe endocytosis pathway - gen

Answer 2

clathrin coated vesicles - no specificity with cargo
like cholesterol most have receptors
early endosome –> late endosome –> endolysosome –> lysosome
all components degraded - macromolecules to monomers

Question 3

describe endosome tethers

Answer 3

complexes have same core subunits
different end subunits bind diff rabs
also bind snares
both recognize hops = bring close to vesicle, also reognized by rab 7 in late endosome
hops = to lysosome, = conformational change so will bind to receptor compartment
to endosomes or to lysosomes

Question 4

name endosome tethers

Answer 4

to endosomes = rab5 and corvet
to lysosomes = rab7 and hops

Question 5

describe rab cascades

Answer 5

Rab5 at endosomes activates Rab7
Rab5 effector and tether (CORVET) is GEF for Rab7
Rab7 effector is GAP to inactivate Rab5
As Rab5 vesicles fuse with early endosome, more and more Rab7 is activated, less and less Rab5 stays active
Membrane becomes late endosome

Question 6

describe clustering of tethers

Answer 6

Rab5 effectors have GEF or PI kinase activity on early endosome
GEF activity produces more Rab5-GTP in local area of membrane
PI-phosphates provide additional binding sites for vesicle tethers
Cluster of tethers form “landing site” for vesicle

Question 7

describe the landing site for vesicle

Answer 7

type of microdomain that does not involve memebrane thickness or lipid content
not thickened but tether rich

Question 8

describe snare proteins

Answer 8

family of membrane proteins that carry out vesicle fusion

Question 9

what can rabs and tethers do

Answer 9

recruits snares to fusion site
membrane of vesicle and receptor compartment

Question 10

describe vesicle snares and target snares - gen function

Answer 10

v snares on vesicles recognize t snares on target membranes
complexes form after tethering - complex provides specificity - where vesicle delivers cargo
unique combos of v and t snares determine targeting specificity

Question 11

describe v snares

Answer 11

monomers with single tm helical domain
integral part
other proteins that interact with other proteins and 2 anchor proteins

Question 12

describe t snares

Answer 12

trimers
combos of TM and peripheral subunits

Question 13

describe what snares do

Answer 13

correct set of v and t snares form a stable tetramer
multiple snare complexes form at a target site to induce vesicle fusion

Question 14

describe snare folding

Answer 14

v snare monomer = no stably folded, passively folded
t snare trimer = partially stable 2 hexlix bundle
v and t snare fold into v stable 4 helix bundle, produces bend and pulls close to target complex
folding process pulls membranes close together = becomes properly folded, generates physical strain like spring, not dependent on atp ot gtp, brings clsoe together and makes it fuse

Question 15

describe fusion model - 4

Answer 15

1. SNARE complexes form in a ring around the vesicle contact site
2. The SNARE TM anchors are bent and strained, exerting a force that holds the membranes together
3. The outer and inner layers of the membranes fuse
4. The strain in the SNARE complex is relieved

Question 16

describe snare dissosciation

Answer 16

After fusion, the SNARE complex is stable, unstrained and inactive
An AAA-family ATPase (NSF) dissociates v- and t-SNAREs – essential for continuation of vesicle traffic
t-SNAREs become active again
v-SNAREs are recycled back to their donor membrane by vesicles

Question 17

describe NSF

Answer 17

NSF binds SNARE complex through adaptor protein (α-SNAP)
NSF twists and pulls during ATP hydrolysis
Multiple cycles of ATPase unwind the SNARE helices

Question 18

describe endocytosis pathway early endosome – lysosome

Answer 18

CCVs mediate endocytosis from PM to early endosome
Some PM proteins are transported to recycling endosome, for exocytosis back to PM
lysosomal proteins are trafficked from trans-Golgi to endosomes, their receptors are trafficked back
Endosome membrane invaginates to form multivesicular body (MVB) and late endosome
Late endosome matures into lysosome, for degradation of proteins and lipids

Question 19

describe receptor recycling - gen

Answer 19

like cholesterol = hydrophobic to protected by carrier
ldl receptors - clathrin coated put, endocytosis and uncoating and fusion with endosome into early endsome - lower ph so receptor unbinds carrier
fuses to lysosome
hydrolytic enzymes for carrier
returns ldl receptors to pm
frees cholesterol

Question 20

describe receptor recycling - specific

Answer 20

Extracellular ligands are bound by transmembrane PM receptors, transported to early endosome for sorting
pH of early endosome is lower than the extracellular space, causing ligands to separate from receptors = changes the average charge on proteins, interactions are weakened
Empty receptors are recycled back to PM
Free ligands progress to lysosome

Question 21

describe retrograde traffic

Answer 21

Retrograde traffic of proteins from endosomes to PM or trans-Golgi= extracellular receptors to PM, receptors that bring proteins to the lysosome are returned to Golgi
involve membrane tubules or tubular vesicle, not round coated vesicles
Requires the Retromer protein complex
does not depend on coated vesicles, but membrane tubules and retromer protein complex

Question 22

describe endosome maturation - mono ub

Answer 22

Some TM proteins are marked for endocytosis by modification with mono-ubiquitin at PM=
– not poly-ubiquitin
– recognition by CCV adaptors
– if ubiquitin is removed, proteins are recycled to PM
– ubiquitin not removed is a signal for lysosomal degradation
tm proteins need to be degraded = if left then signals things, to degrade = early endosome makes another vesicle to grab receptor and bring inside vesicle, = multivesicular body, since cannot face cytosol

Question 23

what turns into mvbs

Answer 23

early endosomes by invaginating and pinching off membrane
mvb contents cannot be recycled into pm anymore

Question 24

describe mvb invagination

Answer 24

a series of ESCRT protein complexes shape and pinch off vesicles into lumen of endomsome

Question 25

name and describe proteins that help mvb formation

Answer 25

ESCRT-0 binds PI(3)P and collects mono-Ub cargo proteins, provides binding site for ESCRT-I
ESCRT-I and II form the neck of the bud
ESCRT-III forms oligomers to pinch off the bud to form vesicle

Question 26

describe lysosomes

Answer 26

MVBs fuse with vesicles containing proteases and other enzymes to become lysosomes
After the ER, the lysosome is the final site of protein degradation in the secretory pathway
Lysosome lumen is highly acidic pH 5 = normal proteins unfold, lysosomal enzymes are most active at low pH
low ph maintained by atp dependent proton pump
final breakdown products are returned to cytosol by small molecule transporters (brings out components needed to make other stuff)

Question 27

name and describe 4 pathways for lysosome degradation

Answer 27

phagocytosis = component of exterior
endocytosis = specific cargo like cholesterol
macropinocytosis = non specific cargo outside cell
autphagy - no space restriction - can be any size, if sop working = recycle them, degradation system

Question 28

describe autophagy - gen

Answer 28

some specificity from receptor on interior of double membrane, expands till closed
makes autophagosome then autolysosome amd makes into monomers

Question 29

describe autophagy - specifics

Answer 29

Large-scale digestion of cytosol and membranes
Upregulated during starvation to release free amino acids (degraded stuff we do not need)
Lipids and proteins are transported through cytosol to form phagophore vesicle
Phagophore grows to enclose contents in an autophagosome= double membrane, large range of sizes
Autophagosome fuses with lysosome to digest contents

Question 30

describe homotypic fusion

Answer 30

In some fusion events, donor and target membranes are the same = fusion of COP-II vesicles into vesicular-tubular cluster that becomes cis-Golgi, re-formation of ER and Golgi after cell division
Both membranes have identical v- and t-SNAREs, already in complexes and inactive
SNAREs must be separated by NSF to allow new fusion

Question 31

what are retromer tubules

Answer 31

Retromer tubules are very different from coated vesicles, but the same steps take place

Question 32

describe retromer - steps 1 and 2

Answer 32

1. Rab5-GTP or Rab7-GTP initiates retromer formation
   2a. Cargo adaptor (Vps26/29/35) binds Rab and selects transmembrane cargo proteins
   2b. Sorting nexin SNX complex binds adaptor and PI(3)P = causes membrane to curve by interacting with lipids, not by forming a rigid coat, diff SNX proteins for golgi or pm traffic
   2c. SNX and adaptor form a complete retromer unit

Question 33

describe tubule formation - steps 2-4

Answer 33

2d. Clusters of retromer shape the membrane into a long tube = retromer does not form a rigid cage, unlikeCOP-I/II or clathrin
3. Dynamin homologs and cytoskeletal motor proteins pinch off the membrane
4. GTP hydrolysis by Rab causes dissociation of retromer complex and uncoating = necessary for fusion with target